The present invention relates to the detection and diagnosis of infection and disease due to the mycobacteria, especially Mycobacterium tuberculosis and other mycobacteria commonly associated with disease in immunocompromised patients including those with acquired immunodeficiency syndrome (AIDS). The present invention is suited for rapid screening of large populations for the presence of M. tuberculosis carriers, as well as diagnosing and monitoring disease or infection in patients who present at healthcare or public health facilities.
Organisms within the genus Mycobacterium include obligate parasites, saprophytes, and opportunistic pathogens. Most species are free-living in soil and water, but for species such as M. tuberculosis and M. leprae, the causative agents of tuberculosis and leprosy respectively, the major ecological niche is the tissue of humans and other warm-blooded animals.
Despite the fact that most mycobacteria do not cause disease, a relatively small group of organisms within the genus is responsible for a large percentage of human morbidity and mortality worldwide. Tuberculosis remains a major global health problem, with nearly one third of the world""s population infected. Indeed, tuberculosis is the leading cause of death due to a single infectious agent. In addition. the World Health Organization estimates that worldwide, there are 8-10 million new cases and over 3 million deaths directly attributed to this disease reported worldwide (A. Kochi, The global tuberculosis situation and the new control strategy of the World Health Organization, Tubercle 72:1-6 [1991]).
M. tuberculosis is exceptionally easily transmitted, as it is carried in airborne particles termed xe2x80x9cdroplet nuclei,xe2x80x9d produced when a patient with active tuberculosis coughs. These particles are from 1-5xcexc in size, and are readily suspended in air currents. Infection occurs when droplet nuclei are inhaled and reach the terminal airways of the new host""s lungs. Usually, the host immune response limits the multiplication and spread of the organism, although some organisms may remain dormant, but viable, for many years post-infection. Individuals infected with M. tuberculosis but without disease, usually have a positive skin test (i.e., with purified protein derivative [PPD]), but are asymptomatic and generally not infectious. However, latently infected individuals have a 10% risk for developing active tuberculosis at some point during their life; the risk is greatest within the first two years post-infection. For HIV-positive individuals, the risk is much greater, with the risk at 10-15% per year for progression to active disease (F. S. Nolte and B. Metchock, xe2x80x9cMycobacterium,xe2x80x9d in Manual of Clinical Microbiology, Sixth Edition, ASM Press: Washington, [1995],pp. 400-437).
Co-infection with human immunodeficiency virus (HIV) and M. tuberculosis has resulted in staggering increases in tuberculosis ratesxe2x80x94as much as 200% in the past 4 years, particularly in impoverished countries with few resources available to control this epidemic. Yet even western industrialized countries have reported increases in tuberculosis rates of from 2 to 14% per year during the past decade (World Health Organization TB Programme, quoted in xe2x80x9cTB: A Global Emergency,xe2x80x9d WHO, 1994). These increases, coupled with the emergence of multi-drug-resistant strains, and the recognition that tuberculosis is one HIV-related opportunistic infection which can be readily transmitted to HIV-uninfected persons, have focused the attention of physicians, researchers, and public health workers on issues related to tuberculosis control, particularly in terms of development of improved vaccines for tuberculosis prevention and improved tests for tuberculosis diagnosis.
In the United States, aggressive approaches to tuberculosis control including isolation of patients in facilities such as sanitoria and the development of drugs effective against M. tuberculosis resulted in a steady decline in the incidence of tuberculosis until about 1985, when the trend reversed and the reports of new tuberculosis cases began to increase. If the trend for the years 1980-1984 is used to calculate the number of expected cases, the Centers for Disease Control and Prevention (CDC) estimated that between 1985-1992, approximately 51,000 excess cases have accumulated (D. E. Snider et al., xe2x80x9cGlobal burden of tuberculosis,xe2x80x9d in B. R. Bloom (ed.), Tuberculosis: Pathogenesis, Protection and Control, American Society for Microbiology, Washington, D.C., [1994], pp. 3-11).
A number of contributory factors are likely to be responsible for the observed increase in tuberculosis cases, including the AIDS pandemic, immigration from areas with high endemicity of tuberculosis, general deterioration of the health care infrastructure, transmission in high-risk environments (e.g., homeless shelters), and the increase in the number of multi-drug resistant M. tuberculosis strains (Nolte and Metchock, supra, at p. 400). Unless the effectiveness and availability of methods and drugs to detect and treat tuberculosis do not substantially improve, it is expected that over 30 million deaths and 90 million new cases of tuberculosis will occur in the years between 1990-2000 (Snider et al., at p. 10).
Although it is the major cause, organisms other than M. tuberculosis are sometimes associated with tuberculosis in humans and other animals. These organisms are included in the xe2x80x9cM. tuberculosis complex,xe2x80x9d which includes M. bovis, M. africanum, and M. microti, as well as M. tuberculosis. M. bovis causes tuberculosis in cattle, humans and other primates, carnivores (e.g., dogs and cats), swine, parrots, and some birds of prey. Human disease is virtually indistinguishable from that caused by M. tuberculosis, and is treated in a similar manner (Nolte and Metchock, supra, at 402). Similarities between M. bovis and M. tuberculosis led to the development of the bacillus of Calmette-Guxc3xa9rin (BCG) an attenuated form of M. bovis, as a vaccine against tuberculosis in many parts of the world (See e.g., W. K. Joklik et al. (eds.), Zinsser Microbiology, 18th ed., Appleton-Century Crofts, Norwalk, Conn., [1984], p. 564).The human health problems associated with M. bovis were largely responsible for the development of methods for the pasteurization of milk and the adoption of compulsory pasteurization in the early 1900s (See, C. O. Thoen, xe2x80x9cTuberculosis in wild and domestic mammals,xe2x80x9d in B. R. Bloom (ed.) Tuberculosis: Pathogenesis, Protection and Control, American Society for Microbiology, Washington, D.C. [1994], pages 157-162). M. africanum has been reported from cases of tuberculosis in tropical Africa. M. microti causes generalized tuberculosis in voles, and produces local lesions in such animals as guinea pigs, rabbits, and calves (Nolte and Metchock, supra, at 402).
Thus, M. tuberculosis is not the only respiratory pathogen of great public health concern, and neither is the M. tuberculosis complex. Recent developments in the taxonomy and study of the mycobacteria have resulted in recognition of M. avium complex (MAC) organisms as the cause of disseminated disease in immunocompromised patients, in particular AIDS patients. The two major species associated with MAC are M. avium and M. intracellulare. However, the MAC includes 28 serovars of these two distinct species, although three additional serovars of M. scrofulaceum (i.e., M. aviumxe2x80x94M. intracellularexe2x80x94M. scrofulaceum complex) were previously included. Within the M. avium species, three subspecies have been proposed, based on phenotypic and genotypic characteristics (M. avium subspecies avium, M. avium subspecies paratuberculosis, and M. avium subspecies silvaticum) (M.-F. Thorel et al., Numerical taxonomy of mycobactin-dependent mycobacteria, emended description of Mycobacterium avium, and description of Mycobacterium avium subsp. avium, subsp. nov. M. avium subsp. paratuberculosis, subsp. nov., and Mycobacterium avium subsp. silvaticum subsp. nov., Int. J. Syst. Bacteriol., 40:254-260 [1990]). As additional information is gathered on the genetic relationships among the mycobacteria, it is likely that changes will occur in the taxonomy of these organisms.
M. avium, an important pathogen of poultry and swine was recognized as a cause of disease in chickens in the late 1800s, but was not recognized as a cause of human disease until 1943 (See, Nolte and Metchock, supra, at 402). M. intracellulare is usually associated with disease in swine and cattle. In addition to its veterinary significance, MAC is an important pathogen of immunocompromised patients, especially those with AIDS. Indeed, disseminated MAC infection is the most common opportunistic infection observed late in the course of HIV disease. It has been reported that the frequency of disease due to MAC rises from 3% per year for individuals with CD4 counts of 100-200/xcexcl to 39% at CD4 counts of  less than 10/xcexcl (S. D. Nightingale et al., xe2x80x9cIncidence of M. avium intracellulare complex bacteraemia in HIV-positive patients,xe2x80x9d J. Infect. Dis., 165:108-25 [1992]). Disease with MAC is characterized by fever, cachexia, hepatic dysfunction, and anemia. As with infection with M. tuberculosis, infection with MAC may promote HIV expression, leading to accelerated HIV disease progression.
Human acquisition of M. avium appears to occur via inhalation or ingestion of fresh water in which bacilli are concentrated (K. L. Fry et al., xe2x80x9cEpidemiology of infection by nontuberculous mycobacteria. VI. Identification and use of epidemiologic markers for studies of Mycobacterium avium, M. intracellulare, and M. scrofulaceum,xe2x80x9d Am. Rev. Respir. Dis., 134:39-43[1986]). It is hypothesized that local infection is followed by hematogenous spread to organs of the reticuloendothelial system. It is here, in bone marrow and lymph nodes, that the number of MAC colony forming units (CFU) ultimately rise to levels several logs higher than are present in blood (D. Peterson et al., xe2x80x9cM. avium complex (MAC) disease in HIV-infected patients is a uniform infection of bone marrow that does not correlate with the level of infection in blood,xe2x80x9d Natl. Conf. Hum. Retrovir. Rel. Pathogens 2:56 [1995]). The observed mycobacteremia apparently represents xe2x80x9cspilloverxe2x80x9d of bacilli from these heavily infected organs. Alternatively, MAC mycobacteremia may occur intermittently, arising from the gastrointestinal tract, without inevitably causing tissue infection.
Recently, our understanding of the mycobacteria, including the recently described species (e.g., M. genavense) associated with disseminated disease in HIV-infected individuals, as well as the potential pathogens M. asiaticum, M. haemophilum, M. malmoense, M. shimoidei, and M. celatum, has greatly increased, largely due to an increased interest in opportunistic pathogens, especially those associated with disease in AIDS patients. Nonetheless, many problems remain unresolved, and reliable, rapid methods are needed for the detection of latent and active mycobacterial infections, especially in the case of AIDS patients.
Treatment of Mycobacterial Disease and Infection
The unique properties of the mycobacterial cell wall, growth rates, and other factors have provided avenues as well as detours in our development of methods for detection and treatment of mycobacterial disease. The mycobacteria characteristically have cell walls with a high lipid content, including waxes such as mycolic acid. The properties of this waxy cell wall provide the xe2x80x9cacid-fastxe2x80x9d nature of the organisms, as once dye is taken into the cells, they are not easily decolorized, even with acid-alcohol. Thus, unlike most organisms, the mycobacteria are said to be xe2x80x9cacid fastxe2x80x9d and are often referred to as xe2x80x9cacid-fast bacillixe2x80x9d or xe2x80x9cAFB.xe2x80x9d
The growth rate of the mycobacteria ranges from slow to very slow, with generation times ranging from two to 24 hours. Most isolates, including M. tuberculosis require long incubation periods (i.e., 4-8 weeks for traditional culture methods) under optimal conditions for growth to be easily visible in vitro. Once the organisms from a primary culture have grown, biochemical and other tests must be done in order to provide an identification. This is an unacceptably long time between sampling and a definitive identification of the organism causing disease in a patient.
The slow growth rate and the pathogenic processes of M. tuberculosis contribute to problems encountered in treating tuberculosis patients. As most antimicrobial drugs work against actively growing cultures, the relatively metabolically inactive mycobacteria enclosed within relatively impermeable waxy cell walls are generally unaffected by most drugs commonly used to combat bacterial disease.
The first line of drugs used against M. tuberculosis include isoniazid (INH), rifampin, pyrazinamide, ethambutol, and streptomycin. The second line drugs include para-amino salicylic acid, ethionamide, cycloserine, capromycin, kanamycin, amikacin, ciprofloxacin, ofloxacin and rifabutin. It is recommended that patients initially be treated with INH, rifampin, ethambutol, and pyrazinamide for 2 months. Those patients with fully drug-susceptible isolates then may be treated with INH and rifampin for an additional four months (American Thoracic Society and Centers for Disease Control, xe2x80x9cTreatment of tuberculosis and adults and children,xe2x80x9d Am. Rev. Respir. Dis., 134:355-363 [1986]). Patients with isolates resistant to either INH or rifampin must be treated with alternative regimens for longer duration. In all cases, successful treatment of patients must continue long after acid-fast bacilli are no longer detected in sputum samples.
Drug resistant M. tuberculosis strains of have become a serious concern worldwide. In a recent nationwide survey of drug resistance among tuberculosis cases reported during the first quarter of 1991, it was found that 14.9% had isolates that were resistant to at least one anti-tuberculosis drug, and 3.3% had isolates resistant to both INH and rifampin (Centers for Disease Control, xe2x80x9cNational MDR-TB Task Force, national action plan to combat multidrug-resistant tuberculosis,xe2x80x9d Morbid. Mortal. Wkly. Rept. 41:1-48 [1992]). This is of grave concern, as INH and rifampin are the most effective drugs in our arsenal against M. tuberculosis. 
For MAC, the concerns are potentially even more significant. Strains of MAC have been reported to be intrinsically resistant to anti-tuberculosis drugs and many other antimicrobials due to failure of these drugs to penetrate the lipid-rich cell wall (N. Rastogi et al., xe2x80x9cEnhancement of drug susceptibility of Mycobacterium avium by inhibitors of cell envelope synthesis,xe2x80x9d Antimicrob. Agents Chemother., 34:759-764 [1990]; and N. Rastogi et al., xe2x80x9cSimplified acetylcysteine-alkali digestion-decontamination procedure for isolation of mycobacteria from clinical specimens,xe2x80x9d J. Clin. Microbiol., 25:1428-1438 [1987]). Indeed, optimal regimens for treatment of either chronic pulmonary disease or disseminated MAC infections in AIDS patients have not been defined. In addition, no therapeutic regimen has been shown to be of sustained clinical benefit for patients with disseminated MAC (Nolte and Metchock, supra, at p. 428). It is recommended that patients with HIV and  less than 100 CD4+ cells be given prophylaxis against MAC that is to be continued for the patient""s life, unless multi-drug therapy becomes necessary due to disseminated disease (Centers for Disease Control, xe2x80x9cRecommendations on prophylaxis and therapy for disseminated Mycobacterium avium complex for adults and adolescents infected with human immunodeficiency virus,xe2x80x9d Morbid. Mortal. Wkly Rept., 42(RR):14-20 [1993]). Preventive therapy for MAC can be problematic because of interactions with other drugs commonly used in HIV infection, particularly the new protease inhibitors. Although no optimal treatment regimen has been defined for disseminated MAC, the U.S. Public Health Service Task Force on Prophylaxis and Therapy for MAC recommends that treatment continued for the lifetime of the patient, even if improvement is noted; this treatment should include at least two chemotherapeutic agents, one of which should be azithromycin or clarithromycin.
The situation is even potentially more grim if other Mycobacterium species are associated with disease in a patient, as almost all of the strains of the xe2x80x9crapid growerxe2x80x9d (ie., in vitro growth may be observed in as few a two days) Mycobacterium species (i.e., M. chelonae, M. fortuitum, M. abscessus, etc.) are resistant to the anti-tuberculosis drugs. Thus, prophylactic treatment of immunocompromised patients is problematic and treatment is dependent upon the results of antimicrobial susceptibility testing of patient isolates.
Detection of Mycobacterial Disease and Infection
The field of diagnostic and clinical microbiology has continued to evolve, and yet, there remains a general need for systems that provide rapid and reliable detection of disease and infection due to microorganisms such as M. tuberculosis. Nontheless, the role of the clinical mycobacteriology laboratories cannot be underestimated in view of their potential contributions in controlling the spread of tuberculosis and non-tuberculosis disease through the timely detection, isolation, identification, and determination of drug susceptibility of these organisms. There is a xe2x80x9cnew sense of urgencyxe2x80x9d regarding the reporting of acid-fast smear, cultures, and drug susceptibility results to physicians, prompted in large part to the emergence of multi-drug resistant strains of M. tuberculosis (Nolte and Metchock, supra, at p. 400). Traditionally, tuberculosis surveillance has involved the use of preliminary skin tests (e.g., tuberculin tests), with positives being further evaluated for active disease by radiographic analysis (i.e., chest X-rays), and sputum cultures. Other samples are sometimes submitted to the laboratory for culture, including blood, bronchoalveolar lavage fluid, bronchial washings, gastric lavage fluids, urine, body fluids (e.g., cerebrospinal [CSF], pleural, peritoneal, pericardial, etc.), tissues (e.g., lymph nodes, skin, or other biopsy materials), abscess contents, aspirated fluids, skin lesions, and wounds.
In contrast to the situation with tuberculosis, blood cultures are often used for the isolation of MAC from immunocompromised patients, especially those with AIDS. Positive MAC blood cultures are generally, but not always, associated with clinical evidence of tissue infection, which typically can involve the bone marrow, liver, or lymph nodes (J. Havlik et al., xe2x80x9cDisseminated Mycobacterium avium complex infection: clinical identification and epidemiologic trends,xe2x80x9d J. Infect. Dis., 165:577-580 [1992], F J. Torriani et al., xe2x80x9cAutopsy findings in AIDS patients with M. avium complex bacteremia,xe2x80x9d J. Infect. Dis., 170:1601-5 [1994]). Thus, detection of MAC in tissue samples provides useful information on a patient""s status. However, culturing of blood remains the most commonly used method for diagnosis of MAC infection, primarily because of the requirement for an invasive procedure to sample infected tissues.
Culture Methods. Once a specimen has been received in the laboratory suspected of containing mycobacteria, the specimen will generally be stained and examined for the presence of AFB. Sputum and other xe2x80x9cdirtyxe2x80x9d specimens are decontaminated and concentrated prior to staining and culturing. Specimens are inoculated onto either solid egg-based media (i.e., Lowenstein-Jensen agar), or liquid medium, in which growth is more rapid. Various commercial growth media systems and methods are available for detection of mycobacteria, including BACTEC (Becton-Dickinson Diagnostic Instrument Systems), Septi-Chek (Becton-Dickinson Microbiology Systems), and the Isolator system (Wampole). Automated detection methods (e.g., those based on production of radiolabelled CO2, turbidity or light) have been developed to identify cultures with microbial growth. However, growth of M. tuberculosis and other mycobacterial species must be confirmed by other methods. Methods presently accepted for detection and identification of are described in great detail in various publications (see e.g., N. Master (section editor) Section 3. xe2x80x9cMycobacteriology,xe2x80x9d in H. E. Isenberg (editor in chief), Clinical Microbiology Procedures Handbook, volume 1, 3.0.1-3.16.4 [1994]).
Immunological Methods. Methods for diagnosis of tuberculosis based on immunologic methods such as detection of delayed type hypersensitivity (DTH) skin responses, as well as the detection of anti-mycobacteria antibodies, or mycobacterial antigens have been studied. Historically, skin tests have been commonly used as indicators of infection with M. tuberculosis. The tuberculin skin test, still commonly in use, was the first immunodiagnostic test developed for detection of tuberculosis. Problems with this test include its inability to distinguish active disease from past sensitization, as well as its unknown predictive accuracy (D. Snider, xe2x80x9cThe tuberculin skin test,xe2x80x9d Am. Rev. Resp. Dis., 125(Suppl.):108-118 [1982]). Even among healthy skin test reactors, the test cannot distinguish those individuals with continued latent infection (in whom there is a continued risk of developing active disease) from those in whom a protective immune response has eradicated that infection. In vitro tests to determine the cell-mediated responses to mycobacterial antigens have also been described. However, they are expensive, technically demanding to perform and interpret, and provide no additional data than are available from skin testing (See e.g., Nolte and Metchock, supra, at p. 426).
Despite the fact that much effort has been devoted to development of serological tests for tuberculosis, these methods have not found widespread clinical use (E. Bardana, xe2x80x9cUniversal occurrence of antibodies to tubercle bacilli in sera from non-tuberculous and tuberculous individuals,xe2x80x9d Clin. Exp. Immunol., 13:65-77 [1973]; and T. M. Daniel and S. M. Debanne, xe2x80x9cThe serodiagnosis of tuberculosis and other mycobacterial disease by enzyme-linked immunosorbent assay,xe2x80x9d Am. Rev. Resp. Dis., 158:678-680 [1987]). Nonetheless, perhaps because antibody detection methods are commonly used in the diagnosis of infectious disease, assay methods based on anti-mycobacterial antibody detection have been investigated.
Antibody Detection. Several studies have focused on methods to determine the anti-mycobacterial antibody level in patients"" sera. These studies have used a variety of antigen preparations, including crude extracts, purified native antigens, and recombinant proteins. Immunoassays, including ELISAs and radioimmunoassays (RIA) have been used in many of these studies (See e.g., E. G. Wilkins et al., xe2x80x9cA Rapid, Simple Enzyme Immunoassay for Detection of Antibody to Individual Epitopes in the Serodiagnosis of Tuberculosis,xe2x80x9d Eur. J. Clin. Microbiol. Infect. Dis., 10: 559-563 [1991]; R. G. Benjamin et aL, xe2x80x9cSerodiagnosis of Tuberculosis Using the Enzyme-Linked Immunoabsorbent Assay (ELISA) of Antibody to Mycobacterium tuberculosis Antigen 51-3,xe2x80x9d Amer. Rev. Respir. Dis., 126:1013-1016 [1982]); R. Maes et al., xe2x80x9cDevelopment of an Enzyme Immunoassay for the Serodiagnostic of Tuberculosis and Mycobacterioses,xe2x80x9d Med. Microbiol. Immunol., 178: 323-335 [1989]); S. B. Kalish et al., xe2x80x9cUse of an Enzyme-Linked Immunosorbent Assay Technique in the Differential Diagnosis of Active Pulmonary Tuberculosis in Humans,xe2x80x9d J. Infect. Dis., 147: 523-530 [1983]); E. Nassau et al., xe2x80x9cThe Detection of Antibodies to Mycobacterium tuberculosis by Microplate Enzyme-Linked Immunosorbent Assay (ELISA),xe2x80x9d Tubercle, 57: 67-70 [1976]; F. L. Garcia-Carreno, xe2x80x9cEnzyme Immunoassay Using BCG in Serodiagnosis of Pulmonary Tuberculosis,xe2x80x9d J. Hyg., 97: 483-487 [1986]; R. Hernandez et al., xe2x80x9cSensitive Enzyme Immunoassay for Early Diagnosis of Tuberculous Meningitis,xe2x80x9d J. Clin. Microbiol., 20:533-535 [1984]; J. A. McDonough et al., xe2x80x9cMicroplate and Dot Immunoassays for the Serodiagnosis of Tuberculosis,xe2x80x9d J. Lab Clin. Med., 120:318-322 [1992]; E. Sada et al., xe2x80x9cAn ELISA for the Serodiagnosis of Tuberculosis Using a 30,00-Da Native Antigen of Mycobacterium tuberculosis,xe2x80x9d J. Infect. Dis., 162:928-931 [1990]; A. Mathai et al., xe2x80x9cRapid Diagnosis of Tuberculous Meningitis with a Dot Enzyme Immunoassay to Detect Antibody in Cerebrospinal Fluid,xe2x80x9d Eur. J. Clin. Microbiol. Infect. Dis., 10:440-443 [1991]; M. Turneer et al., xe2x80x9cHumoral Immune Response in Human Tuberculosis: Immunoglobulins G, A, and M Directed against the Purified P32 Protein Antigen of Mycobacterium bovis Bacillus Calmette-Guerin,xe2x80x9d J. Clin. Microbiol., 26: 1714-1719 [1988]; N. K. Kaushik et al., xe2x80x9cSerodiagnostic Efficiency of Phospholipid Associated Protein of Mycobacterium tuberculosis H37Rv,xe2x80x9d Med. Microbiol. Immunol., 182:317-327 [1993]; D. Kumar et al., xe2x80x9cIdentification of a 25-Kilodalton Protein of Mycobacterium bovis BCG to Distinguish BCG Strains from Mycobacterium tuberculosis,xe2x80x9d J. Clin. Microbiol., 34: 224-226 [1996]; Chandramuki et al. xe2x80x9cLevels of Antibody to Defined Antigens of Mycobacterium tuberculosis in Tuberculous Meningitis,xe2x80x9d J. Clin. Microbiol., 27: 821-825 [1989]; K. A. Near et al., xe2x80x9cUse of Serum Antibody and Lysozyme Levels for Diagnosis of Leprosy and Tuberculosis,xe2x80x9d J. Clin. Microbiol., 30: 1105-1110 [1992]; and H. Mixc3x6ner et al., xe2x80x9cDiagnosis of Tuberculous Meningitis: A Comparative Analysis of 3 Immunoassays, An Immune Complex Assay and the Polymerase Chain Reaction,xe2x80x9d Tubercle Lung Dis., 76: 381-386 [1995].
Although as listed above, numerous researchers have attempted to develop immunodiagnostic systems based on detection of antibody directed against mycobacterial antigens, no antibody tests have been accepted or sufficiently developed for routine diagnosis of tuberculosis. This is in large part due to the fact that the specificities of tests that use crude antigens are too low to be useful clinically. In addition, not all patients respond to the same mycobacterial antigens; any increased specificity achieved by using purified antigens is compromised by a concomitant decrease in sensitivity (See e.g., P. S. Jackett et al., xe2x80x9cSpecificity of Antibodies to Immunodominant Mycobacterial Antigens in Pulmonary Tuberculosis,xe2x80x9d J. Clin.Microbiol., 26: 2313-2318 [1988]). Lastly, immune responses are inadequate in immunocompromised hosts who are at greatest risk of developing tuberculosis. In order to avoid the problems associated with detecting host immune response, detection methods for mycobacterial antigens themselves have been investigated.
Antigen Detection. Detection of microbial antigens in fluids remote from the site of infection has been applied to diagnosis and monitoring of therapy for several infectious diseases other than tuberculosis, including cryptococcosis, histoplasmosis, and, on an experimental basis, leprosy. The type of antigen and the optimal strategy for testing varies according to the illness. In the case of cryptocococcis, a polysaccharide capsular antigen can be detected in cerebrospinal fluid and blood using a simple latex agglutination test (A. A. Gal et al., xe2x80x9cThe clinical laboratory evaluation of cryptococcal infections in the acquired immunodeficiency syndrome,xe2x80x9d Diagn. Microbiol. Infect. Dis., 7:249-54 [1987]). In disseminated histoplasmosis, a heat stable polysaccharide antigen may be detected in blood, CSF, and urine by radioimmunoassay (L. J. Wheat et al., xe2x80x9cDiagnosis of disseminated histoplasmosis by detection of Histoplasma capsulatum antigen in serum and urine specimens,xe2x80x9d N. Engl. J. Med., 314:83-8 [1986]). In leprosy, serum levels of M. leprae phenolic glycolipid I correlate with bacillary load at diagnosis and during therapy, ranging from 12 ng/ml in paucibacillary patients to as high as 8000 ng/ml in multibacillary patients (D. B. Young et al., xe2x80x9cDetection of phenolic glycolipid I in sera from patients with lepromatous leprosy,xe2x80x9d J. Infect. Dis., 152:1078-81 [1985]). However, the successes with these organisms have not been mirrored in diagnosis of tuberculosis and MAC disease, with the exception of one report for MAC, described below.
Detection of Mycobacterial Antigens. Previous reports of antigen detection assays for rapid diagnosis of tuberculosis have been limited to examination of fluids obtained from the site of clinical disease, such as cerebrospinal fluid, sputum, or bronchoalveolar lavage fluid (E. Sada et al., xe2x80x9cDetection of mycobacterial antigens in cerebrospinal fluid of patients with tuberculous meningitis by enzyme-linked immunosorbent assay,xe2x80x9d Lancet 2:651-2 [1983]; I. O. al Orainey et al., xe2x80x9cDetection of mycobacterial antigens in sputum by an enzyme immunoassay,xe2x80x9d Eur. J. Clin. Microbiol. Infect. Dis., 11:58-61 [1992]; R. Kansal et al., xe2x80x9cDetection of mannophosphoinositide antigens in sputum of tuberculosis patients by dot enzyme immunoassay,xe2x80x9d Med. Microbiol. Immunol. Berl., 180:73-8 [1991]; M. A. Yanez et al., xe2x80x9cDetermination of mycobacterial antigens in sputum by enzyme immunoassay,xe2x80x9d J. Clin. Microbiol., 23:822-5 [1986]; G. V. Kadival et al., xe2x80x9cRadioimmunoassay of tuberculous antigen,xe2x80x9d Indian J. Med. Res., 75:765-70 [1982], A. Chandramuki et al., xe2x80x9cDetection of mycobacterial antigen and antibodies in the cerebrospinal fluid of patients with tuberculous meningitis,xe2x80x9d J. Med. Microbiol., 20: 239-247 [1985]; C. L. Cambiaso et al., xe2x80x9cImmunological detection of mycobacterial antigens in infected fluids, cells and tissues by latex agglutinationxe2x80x94Animal model and clinical application,xe2x80x9d J. Immunol. Meth., 129: 9-14 [1990]). A number of other antigen assays have also been described (See e.g., T. M. Daniel, xe2x80x9cRapid diagnosis of tuberculosis: Laboratory techniques applicable in developing countries,xe2x80x9d Rev. Infect. Dis., 2(Supplement 2): S471-S478 ([1989]).
In other studies, the infected fluids were placed in liquid culture for a short period, and mycobacterial products were detected in the culture medium (A. Raja et al., xe2x80x9cSpecific detection of Mycobacterium tuberculosis in radiometric cultures by using an immunoassay for antigen 5,xe2x80x9d J. Infect. Dis., 158:468-70 [1988]; A. Raja et al., xe2x80x9cThe detection by immunoassay of antibody to mycobacterial antigens and mycobacterial antigens in bronchoalveolar lavage fluid from patients with tuberculosis and control subjects,xe2x80x9d Chest 94: 133-137 [1988]; R. Schoningh R et al., xe2x80x9cEnzyme immunoassay for identification of heat-killed mycobacteria belonging to the Mycobacterium tuberculosis and Mycobacterium avium complexes and derived from early cultures,xe2x80x9d J. Clin. Microbiol., 28:708-13 [1990]; A. Drowart et al., xe2x80x9cDetection of mycobacterial antigens present in short-term culture media using particle counting immunoassay,xe2x80x9d Am. Rev. Respir. Dis., 147:1401-6 [1993]). The detection threshold of these assays ranged from 1 ng to 1 xcexcg/ml. All used a polyclonal antiserum rather than monoclonal antibody for capture; several used the same serum for both capture and detection. In none of these reports were fluids remote from the site of infection studied, and in none could the assay identify subjects with latent infection.
M. tuberculosis Antigens. In contrast to the situation with many bacteria, the antigens of M. tuberculosis are a remarkably complex mixture of proteins, polysaccharides, and lipids. The polysaccharide antigens share antigenic cross-reactivity with Nocardia, the corynebacteria, and staphylococci, and generally do not elicit delayed type hypersensitivity (DTH) (Y. Yamamura et al., xe2x80x9cBiology of the mycobacterioses. Chemical and immunological studies on peptides and polysaccharides from tubercle bacilli,xe2x80x9d Ann. NY Acad. Sci., 154:88-97 [1968]; and S. D. Chaparas et al., xe2x80x9cComparison of lymphocyte transformation, inhibition of macrophage migration and skin tests using dialyzable and nondialyzable tuberculin fractions from Mycobacterium bovis (BCG),xe2x80x9d J. Immunol., 107:149-53 [1971]). However, the protein antigens of the Mycobacterium elicit a DTH response, and stimulate lymphocyte blastogenic responses in both sensitized humans and guinea pigs (L. F. Affronti et al., xe2x80x9cSome early investigations of Mycobacterium tuberculosis,xe2x80x9d Am. Rev. Respir. Dis., 92:1-8 [1995]); T. M. Daniel et al., xe2x80x9cReactivity of purified proteins and polysaccharides from Mycobacterium tuberculosis in delayed skin test and cultured lymphocyte mitogenesis assays,xe2x80x9d Infect. Immun., 9:44-7 [1974]; and S. D. Chaparas et al., xe2x80x9cTuberculin-active carbohydrate that induces inhibition of macrophage migration but not lymphocyte transformation,xe2x80x9d Science 170:637-9 [1970]).
The antigenic repertoire of M. tuberculosis includes some proteins which are also found intracellularly, and some which appear uniquely as secreted proteins. As the technology to define these antigens has improved, their number has grown. In 1971, 11 antigens could be identified by immunoprecipitation in culture filtrate (the spent medium of cultures of M. tuberculosis after the bacilli have been removed by filtration (B. W. Janicki et al., xe2x80x9cA reference system for antigens of Mycobacterium tuberculosis,xe2x80x9d Am. Rev. Respir. Dis., 104:602-4 [1971]). Antigen 5 (a 38 kD protein), and antigen 6 (a 30-32 kD protein later termied alpha antigen, BCG 85B, and MPT59), were prominent, and appeared to have some antigenic determinants which were restricted to M. tuberculosis (T. M. Daniel et al., xe2x80x9cImmunobiology and species distribution of Mycobacterium tuberculosis antigen 5,xe2x80x9d Infect. Immun., 24:77-82 [1979]; T. M. Daniel et al., xe2x80x9cDemonstration of a shared epitope among mycobacterial antigens using a monoclonal antibody,xe2x80x9d Clin. Exp. Immunol., 60:249-58 [1985]; and T. M. Daniel et al., xe2x80x9cSpecificity of Mycobacterium tuberculosis antigen 5 determined with mouse monoclonal antibodies,xe2x80x9d Infect. Immun., 45:52-5 [1984]). A decade later, Closs and colleagues identified as many as 50 distinct antigens using a system of crossed immunoelectrophoresis (O. Closs et al., xe2x80x9cThe antigens of Mycobacterium bovis, strain BCG, studied by crossed immunoelectrophoresis: a reference system,xe2x80x9d Scand. J. Immunol., 12:249-63 [1980]). The use of 2-D gel electrophoresis has increased this number to greater than 100. This growing number of potential antigens has presented a challenge in the development of diagnostic and treatment systems.
MAC Antigens. In contrast to these reports in which only infected fluids were studied, a method was reported for detection of a MAC protein antigen in urine of AIDS patients with disseminated MAC infection (A. A. Sippola et al., xe2x80x9cMycobacterium avium antigenuria in patients with AIDS and disseminated M. avium disease,xe2x80x9d J. Infect. Dis., 168:466-8 [1993]). This assay utilized a goat antiserum (xe2x80x9cK-IIxe2x80x9d) which had been developed by immunization with M. intracellulare, and which primarily recognized a 22.5 kD antigen of M. intracellulare, M. avium and M. scrofulaceum, but not M. tuberculosis. Antigenuria was detected in clinical specimens using an assay in which supported nitrocellulose strips were dipped into voided urine samples, which were then probed with the antiserum. Antigenuria was detected in 7/11 patients with M. avium-complex disease, and in 16/100 HIV+ controls. Two of the apparent false positive controls were subsequently found to have disseminated M. avium infection.
Although this assay format was initially appealing in terms of its simplicity, it has several major shortcomings, including: the use of supported nitrocellulose strips hinders uniform washing of multiple samples; the binding of the target antigen to the paper is non-specific; the assay cannot be used to detect non-protein antigens, as their binding to the paper may not be adequate. Also, the specificity of the assay is entirely determined by that of the antiserum. It is therefore critical that the serum not cross-react with human proteins or antigens of other pathogens, an unrealistic expectation for this assay. Therefore, the advantages of these researchers"" assay may be outweighed by its disadvantages and/or be inadequate to offer clinicians a substantial advantage when compared to the use of blood cultures for detection of mycobacteria.
Effect of HIV Infection on Diagnosis of Mycobacterial Disease
HIV infection alters the manifestations of tuberculosis, particularly in those patients with advanced HIV disease, in whom half have mycobacteremia and more than 75% have lymph node, hepatic, or bone marrow involvement, particularly those with low CD4 counts (B. E. Jones et al., xe2x80x9cRelationship of the manifestations of tuberculosis to CD4 cell counts in patients with human immunodeficiency virus infection,xe2x80x9d Am. Rev. Respir. Dis., 148:1292-1297 [1993]; G. I. Santos et al., xe2x80x9cLiver disease in patients with human immunodeficiency virus infection. Study of 100 biopsies,xe2x80x9d Rev. Clin. Esp., 193:115-8 [1993]; A. D. Pithie et al., xe2x80x9cFine-needle extrathoracic lymph-node aspiration in HIV-associated sputum-negative tuberculosis,xe2x80x9d Lancet 340:1504-5 [1992]; and P. F. Barnes et al., xe2x80x9cTuberculosis in the 1990s,xe2x80x9d Ann. Intern. Med., 119:400-10 [1993].
However, HIV infection is accompanied by less radiographic evidence of pulmonary disease, fewer lung zones with cavitation, and reduced numbers of mycobacterial colony-forming units in sputum (R. J. Brindle et a., xe2x80x9cQuantitative bacillary response to treatment in HIV-associated pulmonary tuberculosis,xe2x80x9d Am. Rev. Respir. Dis., 147:958-61 [1993]). Thus, the likelihood of diagnosis based on expectorated sputum is reduced in HIV-associated tuberculosis, even though the total mycobacterial burden may be greater. This has led to the necessity of increased evaluation of specimens other than sputum, particularly tissues such as pleura, liver, and bone marrow. Patients with miliary or disseminated tuberculosis, particularly those with HIV infection, often require multiple biopsies prior to initiation of therapy.
Again, despite the large number of studies, no antigen detection method has been developed to date which provides reliable, highly specific, and highly sensitive results, especially for xe2x80x9cdirtyxe2x80x9d samples such as sputum (See e.g., J. M. Grange, xe2x80x9cThe rapid diagnosis of paucibacillary tuberculosis,xe2x80x9d Tubercle, 70:1-4 [1989]; and Nolte and Metchock, supra, at p. 426).
Importantly, serodiagnostic tests for tuberculosis, although potentially simple and inexpensive, have been especially hampered by poor sensitivity in HIV-infected persons in whom antibody responses are diminished (T. M. Daniel et al., xe2x80x9cReduced sensitivity of tuberculosis serodiagnosis in patients with AIDS in Uganda,xe2x80x9d Tuber. Lung Dis., 75:33-7 [1994]). This is important because the patient populations that are dually infected with HIV and M. tuberculosis is at greatest risk for developing active tuberculosis (M. E. Villarino et al., xe2x80x9cManagement of persons exposed to multidrug-resistant tuberculosis,xe2x80x9d Morb. Mort. Wkly. Rep., 41(RR-11):61-71 [1992]).
Diagnosis of Latent Infection with M. tuberculosis 
Identification of individuals with latent M. tuberculosis infection is a problem which cannot be addressed by current methods. The natural history of M. tuberculosis infection is such that only 5-10% of individuals who are otherwise healthy will ever develop tuberculosis. As mentioned above, the risk of tuberculosis is highest in the first year following infection, but tuberculosis can occur as long as 50 years later. Thus, the infection clearly is contained but not eradicated in many infected but healthy individuals. Presently, there is no reliable method to distinguish between individuals who are latently infected and those whose infections have been eradicated by a protective immune response. Both groups have positive skin tests with purified protein derivative (PPD), as the longevity of a positive skin test can reflect immunologic memory as well as persistent latent infection.
The presence of immunosuppressive concurrent illnesses increases the likelihood of recrudescent disease in tuberculosis-infected persons. This is most strongly expressed in HIV-co-infected persons, in whom the risk of re-activation of tuberculosis may be increased from 10 to 100 fold. Indeed, HIV has emerged as the most significant risk factor for the progression of latent tuberculosis to active disease (Snider et al., at p. 5; Selwyn et al., xe2x80x9cA prospective study of the risk of tuberculosis among intravenous drug abusers with human immunodeficiency virus infection,xe2x80x9d N. Eng. J. Med., 320:545-550 [1989]). However, skin testing becomes increasingly unreliable as an indicator of M. tuberculosis infection as the CD4 count declines. It thus remains difficult to efficiently target those individuals who would benefit most from preventive therapy.
Yet, identification of latently-infected persons is desirable for initiation of tuberculosis preventive therapy. Although such therapy (daily doses of INH for nine months) can be administered to all skin test positive individuals, such therapy obviously would only benefit those subjects who ultimately would have developed tuberculosis. Thus at least 10-20 subjects must be treated to prevent one case of tuberculosis. The other 9-19 treated subjects are subjected to risks associated with INH (e.g., hepatitis, neuropathy) without any potential benefit. This problem is compounded by the present inability to monitor the effectiveness of preventive therapy. Some individuals will fail preventive therapy because of poor compliance with longterm administration of INH. Others will fail because they were infected with an INH-resistant isolate. At present, there is no method to determine whether preventive therapy has been successful in completely eradicating latent infection with M. tuberculosis. 
In sum, there is no entirely satisfactory method for diagnosis either active tuberculosis or latent mycobacterial infection. Direct examination of sputum or infected tissues is insufficiently sensitive (See e.g., L. B. Heifets and R. C. Good, xe2x80x9cCurrent laboratory methods for the diagnosis of tuberculosis,xe2x80x9d in B. R. Bloom (ed.) Tuberculosis: Pathogenesis, Protection and Control, American Society for Microbiology, Washington, D.C. [1994], pages 85-110). Traditional methods, including cultivation of the organism require the time and facilities for prolonged incubation. Assays based on DNA amplification are expensive and technically demanding, and may not be applicable for routine use in clinical laboratories outside of major medical centers in industrialized countries. The development of a simple, rapid, diagnostic test which does not rely on the growth of organisms in vitro, but that is capable of identifying individuals with latent, subclinical M. tuberculosis infection, and which might predict the likelihood of subsequent relapse, would be of tremendous value for tuberculosis control programs worldwide.
Monitoring of Therapy
Many factors can adversely affect the response to anti-tuberculous therapy. These include primary drug resistance, patient non-compliance, malabsorption, adverse interactions with other medications, and other host factors. Inadequate treatment can lead to emergence of secondary drug resistance due to selective pressures on mycobacterial growth. In order to assess a patient""s response to anti-tuberculosis therapy, patients must be monitored throughout their treatment regimen.
However, sputum cultures and AFB smears return to negative slowly during therapy, such that the proportion of samples which become negative are typically only 40% at 1 month, 80% at 2 months, and 90-95% at 3 months. Chest radiographs also improve slowly, and may not significantly improve until 3 months of treatment. It thus is difficult to identify promptly those patients who ultimately will fail any given tuberculosis treatment regimen. Better early indicators of success or failure clearly are needed.
Mitchison has suggested that the early bactericidal activity of anti-tuberculosis regimens, as determined by quantitative sputum cultures, might predict the overall effectiveness of a treatment regimen (S. L. Chan et al., xe2x80x9cThe early bactericidal activity of rifabutin measured by sputum viable counts in Hong Kong patients with pulmonary tuberculosis,xe2x80x9d Tubercle 1992;33-8 [1992]; and A. J. Jindani et al., xe2x80x9cThe Early Bactericidal Activity of Drugs in Patients with Pulmonary Tuberculosis,xe2x80x9d Am. Rev. Respir. Dis., 121:939-49 [1980]). He observed approximately a 10xe2x88x923 drop in the number of viable M. tuberculosis bacilli during the first 2 weeks of effective multi-drug therapy, and noted lesser reductions with less effective regimens. He suggested that new drugs for tuberculosis might be evaluated in short term studies (1-2 weeks) using quantitative culture as an endpoint. However, this approach has not been widely accepted, largely because of difficulties of performing the assay in a standardized fashion, particularly with regard to homogenization of non-uniform sputum specimens. This lack of standardization precludes the use of this method in the clinical setting.
Problems also exist in monitoring therapy for MAC infection in AIDS patients through serial blood cultures. An autopsy series of 44 patients with MAC bacteremia found that 13 (30%) had no histologic evidence of MAC disease (F. J. Torriani et al., xe2x80x9cAutopsy findings in AIDS patients with M. avium complex bacteremia,xe2x80x9d J. Infect. Dis., 170:1601-5 [1994]), suggesting that transient bloodstream infection may occur and may be self-limited. Conversely, other patients have only transiently or intermittently positive blood cultures in the face of high tissue burdens of mycobacteria (C. A. Kemper et al., xe2x80x9cTransient bacteremia due to M. avium complex in patients with AIDS,xe2x80x9d J. Infect. Dis., 170:488-93 [1994]). These observations suggest that sustained mycobacteremia may be a late event in the natural history of MAC infection, and that blood culture is not an adequate diagnostic or monitoring tool when used alone. However, access to the main site of infection (bone marrow, lymph node, or liver) requires an invasive diagnostic procedure which is not usually undertaken without prior attempts at diagnosis by non-invasive measures. These combined factors often lead to a delay in diagnosis and initiation of therapy, and make it difficult to evaluate the response to therapy.
In sum, despite advances in the detection of M. tuberculosis and other mycobacteria, the need remains for a safe, reliable, easy-to-use system for the detection of infection with these organisms, as well as means for monitoring patients with disease or infection. In particular, there is an urgent need for useful methods to use samples such as urine and other fluids for the detection of infection and disease with Mycobacterium species.
The present invention provides a rapid method for the detection of disease and infection due to the mycobacteria, in particular M. tuberculosis, as well as MAC. The present invention is intended for detection of pulmonary mycobacterial disease or infection, and disseminated mycobacterial disease, as well as localized infection with mycobacteria at sites other than the pulmonary area.
The present invention provides kits for the detection of Mycobacterium in a test sample, comprising: a) a solid support; and b) a monoclonal antibody directed against a portion of alpha antigen immobilized on the solid support. In one embodiment, the kit further comprises a primary antibody, while in another embodiment the kit comprises a reporter antibody, in yet another embodiment the kit further comprises an amplifier system. It is also contemplated that the kit of the present invention comprise a primary antibody and reporter antibody, as well as an amplifier system. In a particularly preferred embodiment, the Mycobacterium species detected is Mycobacterium tuberculosis. In an alternative embodiment the Mycobacterium species detected is Mycobacterium avium. 
It is also contemplated that the kit of the present invention will include additional components, including, but not limited to, such items as an alpha antigen control, a diluent such as saline or water, as well as a plurality of alpha antigen samples with known concentrations of alpha antigen suitable for use in preparing a standard curve(s) for the determination of antigen concentration in the test (i.e., patient) samples. Furthermore, in kits in which biotinylated antibodies are used, in one preferred embodiment, the primary antibody is preadsorbed with an avidin compound (i.e., strepavidin) coupled to biotinylated capture antibody, prior to use in the kit. It is further contemplated that the kit of the present invention comprise methods for analyzing samples using blots, including but not limited to Western blots. These blotting kits may include additional reagents such as those listed above, as well as reagents, including but not limited to, such as paper suitable for the blotting system used, and blocking solution.
It is also contemplated that the antibodies of the kit of the present invention be prepared through the use of synthetic peptides as immunogens. In this embodiment, synthetic peptides of known sequence are used to induce the production of at least one of the antibodies used in the kit. It is also contemplated that the synthetic peptides be used as an immunogen while they are still attached to the beads used to prepare them. Thus, the present invention encompasses antigens of mycobacteria that are naturally occurring and harvested from samples such as sputum, urine or blood samples, as well as mycobacterial antigens that are produced synthetically for use in the production of antibodies for use in the kit. It is also contemplated that these synthetic peptides be used as antigens in the kit. In a preferred embodiment, the synthetic peptides correspond to the alpha antigen of M. tuberculosis or MAC. Thus, the sequences of SEQ ID NOS:1-8 may be used in the form of synthetic peptides for use in the present invention. It is also contemplated that immune complexes will be tested using the kit of the present invention. In this embodiment, the immune complexes may be treated using methods known in the art to dissociate antibodies from antigens.
It is also contemplated that the samples tested using the kit of the present invention will be obtained from individuals infected with one or more types of the human immunodeficiency virus. It is also contemplated that the kit will be used for monitoring the progression of therapy in individuals infected with Mycobacterium, in particular M. tuberculosis and/or MAC. It is further contemplated that the kit will be used with samples from patients who are infected with M. tuberculosis, as determined by skin test positivity, chest radiograph positivity, and/or sputum cultures containing M. tuberculosis. 
The present invention also provides methods for the detection of Mycobacterium in a sample comprising: a) providing: i) a sample suspected of containing at least a portion of the alpha antigen of Mycobacterium and ii) a monoclonal antibody directed against the portion of Mycobacterium alpha antigen; b) adding the sample to the monoclonal antibody under conditions such that the antibody binds to the Mycobacterium alpha antigen in the sample to form an antibody-antigen complex; and c) detecting the binding of said antigen and antibody.
In a preferred embodiment of the method of the present invention, the detecting comprises adding the primary antibody to the antigen-antibody complex so that the primary antibody binds to the antigen to form an antibody-antigen-antibody sandwich. In another preferred embodiment of the method, the detecting further comprises adding a reporter reagent to the antibody-antigen-antibody sandwich to form an antibody-antigen-antibody-antibody sandwich. In a particularly preferred embodiment, the detecting further comprises adding an amplifier to said antibody-antigen-antibody-antibody sandwich.
In one embodiment, the monoclonal antibody comprises a murine monoclonal antibody. In a preferred embodiment, the murine monoclonal antibody is biotinylated. In a particularly preferred embodiment, the primary antibody is preadsorbed with avidin (e.g., streptavidin) coupled to biotinylated capture antibody, prior to use in the method of the present invention for the detection of Mycobacterium species. In yet another particularly preferred embodiment, the solid support is coated with avidin.
In another embodiment, detection is achieved through use of such methods as enzyme immunoassay, radioimmunoassay, fluorescence immunoassay, flocculation, particle agglutination, and in situ chromogenic assay. It is not intended that the present invention be limited to any particular assay format.
It is also contemplated that the method of the present invention will include additional components, including, but not limited to, such items as an alpha antigen control, a diluent such as saline or water, as well as a plurality of alpha antigen samples with known concentrations of alpha antigen suitable for use in preparing standard curve(s) for the determination of antigen concentration in the test (i.e., patient) samples. Furthermore, in methods in which biotinylated antibodies are used, in a preferred embodiment, the primary antibody is preadsorbed with an avidin compound (i.e., strepavidin) coupled to biotinylated capture antibody prior to use in the method. It is further contemplated that the method of the present invention comprise methods for analyzing samples using blots, including but not limited to Western blots. These blotting methods may include additional reagents such as those listed above, as well as reagents, including but not limited to, such as paper suitable for the blotting system used, and blocking solution.
It is also contemplated that the antibodies of the method of the present invention be prepared through the use of synthetic peptides as immunogens. In this embodiment, synthetic peptides of known sequence are used to induce the production of at least one of the antibodies used in the method. It is also contemplated that the synthetic peptides be used as an immunogen while they are still attached to the beads used to prepare them. Thus, the present invention encompasses antigens of mycobacteria that are naturally occurring and harvested from samples such as sputum, urine or blood samples, as well as mycobacterial antigens that are produced synthetically for use in the production of antibodies for use in the method. It is also contemplated that these synthetic peptides be used as antigens in the kit. In a preferred embodiment, the synthetic peptides correspond to the alpha antigen of M. tuberculosis or MAC. Thus, the sequences of SEQ ID NOS:1-8 may be used in the form of synthetic peptides for use in the present invention.
In one embodiment of the method, the Mycobacterium detected by the method of the present invention is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium intracellulare. In one embodiment of the methods of the present invention, the portion of said Mycobacterium alpha antigen is selected from the group comprising SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 and 8. Thus, it is contemplated that fragments of the alpha antigen, as well as the entire alpha antigen will be used in the methods of the present invention. It is also contemplated that immune complexes will be tested using the methods of the present invention. In this embodiment, the immune complexes may be treated using methods known in the art to dissociate antibodies from antigens.
It is contemplated that any sample will be used in the method of the present invention, including but not limited to urine samples, sputum samples, blood samples, and serum samples. In the case of sputum samples, it is contemplated that most samples will be digested and/or decontaminated using standard methods prior to their analysis in the method of the present invention. It is also contemplated that some samples tested using the method of the present invention will be obtained from individuals infected with human immunodeficiency virus, including, but not limited to, HIV-1 and HIV-2.
It is also contemplated that the methods of the present invention will be used for monitoring the progression of therapy in individuals infected with Mycobacterium, in particular M. tuberculosis and/or MAC. It is further contemplated that the methods will be used with samples from patients who are infected with M. tuberculosis, as determined by skin test positivity, chest radiograph positivity, and/or sputum cultures containing M. tuberculosis. 
The present invention also provides methods for the detection of mycobacterial antigen in a urine sample comprising a) providing: i) a urine sample suspected of containing at least a portion of alpha antigen of Mycobacterium; and ii) a monoclonal antibody directed against the portion of Mycobacterium alpha antigen; b) adding the urine sample to the monoclonal antibody under conditions such that the antibody binds to the Mycobacterium alpha antigen in the urine sample to form an antibody-antigen complex; and c) detecting the binding.
The present invention also provides methods for the detection of Mycobacterium in a sample comprising: a) providing: i) a sample suspected of containing at least a portion of the alpha antigen of Mycobacterium and ii) a monoclonal antibody directed against the portion of Mycobacterium alpha antigen; b) adding the sample to the monoclonal antibody under conditions such that the antibody binds to the Mycobacterium alpha antigen in the sample to form an antibody-antigen complex; and c) detecting the binding of said antigen and antibody.
In a preferred embodiment of the method of the present invention, the detecting comprises adding the primary antibody to the antigen-antibody complex so that the primary antibody binds to the antigen to form an antibody-antigen-antibody sandwich. In another preferred embodiment of the method, the detecting further comprises adding a reporter reagent to the antibody-antigen-antibody sandwich to form an antibody-antigen-antibody-antibody sandwich. In a particularly preferred embodiment, the detecting further comprises adding an amplifier to said antibody-antigen-antibody-antibody sandwich.
In one embodiment, the monoclonal antibody comprises a murine monoclonal antibody. In a preferred embodiment, the murine monoclonal antibody is biotinylated. In a particularly preferred embodiment, the primary antibody is preadsorbed with avidin (e.g., streptavidin) coupled to biotinylated capture antibody prior to use in the method of the present invention for the detection of Mycobacterium species. In yet another particularly preferred embodiment, the solid support is coated with avidin.
In another embodiment, detection is achieved through use of such methods as enzyme immunoassay, radioimmunoassay, fluorescence immunoassay, flocculation, particle agglutination, and in situ chromogenic assay. It is not intended that the present invention be limited to any particular assay format.
It is also contemplated that the method of the present invention will include additional components, including, but not limited to, such items as an alpha antigen control, a diluent such as saline or water, as well as a plurality of alpha antigen samples with known concentrations of alpha antigen suitable for use in preparing standard curve(s) for the determination of antigen concentration in the test (i.e., patient) samples. Furthermore, in methods in which biotinylated antibodies are used, in a preferred embodiment, the primary antibody is preadsorbed with an avidin compound (i.e., strepavidin) coupled to biotinylated capture antibody prior to use in the method. It is further contemplated that the method of the present invention comprise methods for analyzing samples using blots, including but not limited to Western blots. These blotting methods may include additional reagents such as those listed above, as well as reagents, including but not limited to, such as paper suitable for the blotting system used, and blocking solution.
It is also contemplated that the antibodies of the method of the present invention be prepared through the use of synthetic peptides as immunogens. In this embodiment, synthetic peptides of known sequence are used to induce the production of at least one of the antibodies used in the method. It is also contemplated that the synthetic peptides be used as an immunogen while they are still attached to the beads used to prepare them. Thus, the present invention encompasses antigens of mycobacteria that are naturally occurring and harvested from samples such as sputum, urine or blood samples, as well as mycobacterial antigens that are produced synthetically for use in the production of antibodies for use in the method. It is also contemplated that these synthetic peptides be used as antigens in the kit. In a preferred embodiment, the synthetic peptides correspond to the alpha antigen of M. tuberculosis or MAC. Thus, the sequences of SEQ ID NOS:1-8 may be used in the form of synthetic peptides for use in the present invention.
In one embodiment of the method, the Mycobacterium detected by the method of the present invention is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium intracellulare. In one embodiment of the methods of the present invention, the portion of said Mycobacterium alpha antigen is selected from the group comprising SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 and 8. Thus, it is contemplated that fragments of the alpha antigen, as well as the entire alpha antigen will be used in the methods of the present invention. It is also contemplated that immune complexes present in urine samples will be tested using the methods of the present invention. In this embodiment, the immune complexes may be treated using methods known in the art to dissociate antibodies from antigens.
It is also contemplated that some samples tested using the method of the present invention will be obtained from individuals infected with human immunodeficiency virus, including, but not limited to, HIV-1 and HIV-2.
It is also contemplated that the methods of the present invention will be used for monitoring the progression of therapy in individuals infected with Mycobacterium, in particular M. tuberculosis and/or MAC. It is further contemplated that the methods will be used with samples from patients who are infected with M. tuberculosis, as determined by skin test positivity, chest radiograph positivity, and/or sputum cultures containing M. tuberculosis. 
The present invention also provides a composition comprising purified monoclonal antibody directed against a portion of Mycobacterium alpha antigen. In one embodiment, the monoclonal antibody is a murine monoclonal antibody. In an alternative embodiment, the murine monoclonal antibody is directed against epitopes of the alpha antigen of M. tuberculosis, M. avium or M. intracellulare. In a particularly preferred embodiment, the murine monoclonal antibody is directed against M. tuberculosis epitopes. In yet another preferred embodiment, the alpha antigen used to produce the murine monoclonal antibody is comprised of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, or 8. It is also contemplated that the monoclonal antibody of the present invention will be produced through the use of synthetic peptides as antigens. In a preferred embodiment, the monoclonal antibody of the present invention is raised against SEQ ID NOS: 3, 4, 6 or 7. It is further contemplated that the monoclonal antibody be produced using any suitable methods for monoclonal antibody production, including intrasplenic methods, cell culture, etc. It is not intended that the monoclonal antibody of the present invention be limited to an antibody produced using a particular method or produced using a particular animal species. It is also not intended that the antibodies used in the present invention be of a particular class of immunoglobulin.